A New Strategy To Improve the Metabolic Stability of Lactone

Sep 26, 2013 - Lactone is a common structural motif in biologically active natural products. However, the metabolic instability of lactone significant...
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A New Strategy to Improve the Metabolic Stability of Lactone: Discovery of (20S, 21S)-21-Fluorocamptothecins as Novel Hydrolytically Stable Topoisomerase I inhibitors Zhenyuan Miao, Lingjian Zhu, Guoqiang Dong, Chunlin Zhuang, Yuelin Wu, Shengzheng Wang, Zizao Guo, Yang Liu, Shanchao Wu, Shiping Zhu, Kun Fang, Jianzhong Yao, Jian Li, Chunquan Sheng, and Wannian Zhang J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Sep 2013 Downloaded from http://pubs.acs.org on September 28, 2013

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

A New Strategy to Improve the Metabolic Stability of Lactone: Discovery of (20S, 21S)-21-Fluorocamptothecins as Novel Hydrolytically Stable Topoisomerase I inhibitors

Zhenyuan Miao, *,† ,§ Lingjian Zhu, † ,§ Guoqiang Dong, † Chunlin Zhuang, † Yuelin Wu, † Shengzheng Wang, † Zizao Guo, † Yang Liu, † Shanchao Wu, † Shiping Zhu, † Kun Fang, † Jianzhong Yao, † Jian Li, ≠ Chunquan Sheng,*, † Wannian Zhang*, †



School of Pharmacy, Second Military Medical University, 325 Guohe Road,

Shanghai 200433, People’s Republic of China ≠

School of Pharmacy, East University of Science and Technology, Shanghai 200237,

P. R. China

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Abstract Lactone is a common structural motif in biologically active natural products. However, metabolically instability of lactone has significantly reduced their in vivo potency. In the present investigation, a new strategy to improve the metabolic stability of lactone was provided by the design of α-fluoroether as a novel bioisostere of lactone. The effectiveness of the α-fluoroether/lactone replacement was validated by the discovery of (20S, 21S)-21-fluorocamptothecins as hydrolytically stable Topoisomerase I inhibitors. A highly potent camptothecin derivative 8l was successfully identified, which showed excellent in vitro and in vivo antitumor activities and represents a promising lead for the discovery of novel antitumor agents. Interestingly, this study also provided new structure-activity relationship for the C21-carbonyl group of camptothecin, which has been regarded as an essential pharmacophore. Our results revealed that the conserved C21-carbonyl group can be replaced by a fluorine substituent. α-Fluoroether may have general application in improving metabolic stability of lactone.

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Introduction Natural products are rich sources of lead compounds in drug discovery.1-4 However, many bioactive

natural products with lactone

(e.g. homoserine lactone5,

camptothecin6 and artemisinin7) tend to be hydrolytic unstable and prone to be converted into ring-opened inactive hydrolyzates under the physiological conditions. It is highly desirable to develop novel bioisosteres of lactone to improve its hydrolytic stability without reducing the biological activity. Fluorine substitution represents an important strategy in drug design because of its high electronegativity and small size.8 Rational introduction of fluorine into bioactive compounds is usually helpful to increase the metabolic stability, adjust the physicochemical properties and enhance the binding affinities.9-13 For example, the replacement of an oxidizable C-H bond by a C-F bond has been frequently employed to increase the metabolic stability. The introduction of a fluorine atom to block the metabolically labile sites has successfully generated several clinical drugs (e.g. ezetimibe14 and celecoxib15). However, the application of fluorine substituent in improving lactone stability has not been explored. Thus, it is interesting to design a fluorine-based new bioisostere of lactone. Herein, camptothecin (CPT, 1, Figure 1) will be used as the model molecule to validate the hypothesis. CPT, a pentacyclic quinoline-based alkaloid isolated from Camptotheca acuminate, exhibits excellent antitumor activity and acts as a topoisomerase I (Top1) inhibitor.6 However, clinical development of CPT was terminated because of its poor aqueous solubility, low metabolic stability of lactone and high in vivo hepatoxicity. Although 3

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subsequent structural modification of natural CPT has generated three antitumor agents (i.e. topotecan, irinotecan and belotecan) and a number of drug candidates, their highly electrophilic α-hydroxylactone of the E ring can be rapidly hydrolyzed to the biologically inactive carboxylate form under the physiological conditions.16-22 Therefore, discovery of novel CPT analogues that can overcome the intrinsic instability has become an active area of research interests.19,

22-24

For example,

homocamptothecin (2)25-30 and α-hydroxy keto-camptothecin (3)31 showed improved metabolic stability.

Figure 1 Chemical structures of the camptothecin derivatives

Due to the important role of fluorine substitution in increasing metabolic stability, herein α-fluoroether was designed as a metabolically stable bioisostere of lactone (Figure 2). As a proof-of-concept study, a series of (20S,21S)-21-fluorocamptothecins were designed and synthesized. Interestingly, these 21-fluorocamptothecin analogues were proven to be potent Top1 inhibitors with increased hydrolytic stability. Moreover, 4

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novel structure-activity relationship (SAR) results were obtained for the CPT-like Top1 inhibitors. The highly active compound 8l showed excellent antitumor activities both in vitro and in vivo, which represent a promising lead for the development of novel antitumor agents. To the best of our knowledge, this is the first of example of using fluorine substitution to improve the metabolic stability of lactone.

Figure 2. Design of α-fluoroether as a hydrolytically stable bioisostere of lactone and its application to improve the metabolic stability of camptothecin derivatives.

Chemistry In our previous studies, diol-CPT 2 can be readily prepared from CPT (1) under KBH4 reduction in a 99% yield (Scheme 1).28 Treatment of 2 with diethylaminosulfur trifluoride (DAST) in methylene dichloride yielded diastereoisomers 5a and 5b and a small amount of 20, 21-difluorocamptothecin. The configurations of compounds 5a (20S, 21R) and 5b (20S, 21S) were assigned by NMR with NOESY experiments according to the proton correlation of H18, H19 and H21 (Figure 3). In order to 5

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investigate the SARs, a series of 21-fluorocamptothecin derivatives (8a-i) with substituents at position 7, 9 or 10 were prepared. Using our established synthetic protocol , compounds 6a-6l were obtained with good yields.28 The replacement of the 21-OH group of compounds 6a-6l by a fluorine was accomplished using similar conditions in Scheme 1. Although the fluorinated CPT derivatives were diastereoisomeric mixtures, the (20S, 21S)-21-fluorocamptothecin derivatives 8a-l could be easily obtained by chromatographic column (Scheme 2). Scheme 1

Reagents and conditions: (a) KBH4, CH3OH, rt, 4h; (b) DAST, CH2Cl2, -78℃-0℃, 2h.

Figure 3. The homo-NOE effects of diastereoisomers 5a and 5b.

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Scheme 2

Reagents and conditions: (a) KBH4, CH3OH, rt, 4h; (b) DAST, CH2Cl2, -78℃-0℃, 2h.

Results and Discussion Design of α-Fluoroether as a Metabolically Stable Bioisostere of Lactone Fluorine substituent has been widely used to block the metabolically labile site in lead optimization. Thus, we envisioned that fluorine could replace the carbonyl group of lactone to afford hydrolytically more stable α-fluoroether. Design rationale of α-fluoroether as a metabolically stable bioisostere of lactone was based on the following considerations. First, the C-F bond has similar electrostatic properties to those of the carbonyl group. Second, the C-F bond can form various protein-ligand interactions that are similar or different to the carbonyl group and thus can influence the binding affinity. For example, the C-F bond can form similar polar (or dipole) interaction with the target protein.32 Although whether fluorine can form hydrogen bonds in biological systems is still controversial, recent studies revealed fluorine 7

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bonding is similar to sulfur-involved hydrogen bonding.33 Moreover, the C-F bond is more lipophilic than the carbonyl group and can form stronger hydrophobic interactions with the target protein. Third, the α-fluoroether substructure can adjust the physicochemical properties (e.g. LogP and pKa) of the molecule and thus modulate the pharmacokinetic behavior. Last, the α-fluoroether substructure is synthetically feasible, which can be easily prepared by reducing and fluorinating the lactone group. In the case of CPT, it should be noted that the C-21 carbonyl group in the E-ring lactone was considered as the essential pharmacophore for the antitumor activity.34 If this C-21 carbonyl group was reduced to a hydroxyl group, namely camptothecin hemilactol (4), the loss of the in vivo antitumor activity was observed.35 Thus, the carbonyl group was always conserved during structural modification of CPT E-ring,34 which makes it difficult to use the α-fluoroether/lactone replacement. However, the crystal structure of CPT-DNA-Top1 ternary complex revealed that the 21-carbonyl group of CPT did not form direct interactions with Top1 and DNA.36 Therefore, 21-fluorocamptothecin derivatives were designed and synthesized to validate the hypothesis. Biological Activity and Solution Stability of 21-Fluorocamptothecin In

order

to

validate

the

designing

rationale,

the

diastereoisomers

of

21-fluorocamptothecin, 5a and 5b, were screened for Top1 inhibitory activity and antiproliferative activity. It is well known that Top1 is the target of CPT, which acts by stabilizing a covalent Top1-DNA complex called the cleavable complex.37 To 8

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investigate whether Top1 is the target of the 21-fluorocamptothecins, Top1-mediated DNA cleavage assays with purified Top1 were performed. Top1 and supercoilded DNA pBR322 were incubated in the presence of the test compounds, whose ability to stabilize the cleavable complex were evaluated by the appearance of short DNA fragments. As shown in Figure 4A, the diastereoisomer 5b was found to be active against Top1-mediated relaxation of supercoiled DNA at the concentration of 100 µM. In contrast, the 21-R-isomer 5a only showed marginal Top1 inhibitory activity.

Figure 4. Top1 and Top 2 inhibitory activity of the fluorinated camptothecin derivatives. (A) Effect of the selected compounds on Topoisomerase-I mediated DNA relaxation in single concentration with 100 µM. Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top1; Lane 3, DNA + Top1 + CPT; Lane 4-9, DNA + Top1 + fluorinated camptothecin derivatives (5a,5b, 8a, 8b, 8h and 8l respectively); Lane 10, DNA + Top1 +6l. (B) Inhibition of Top 2 relaxation activity at 100 µM. Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top 2; Lane 3, DNA + Top 2 + Doxorubicin (DOX); Lane 4, DNA + Top 2 + etoposide (VP 16); Lanes 5, DNA + Top 2 + 8l. (C) Top1-mediated DNA cleavage assay gel of test compounds using supercoiled pUC19 at 100 µM. Lane 1, supercoiled plasmid DNA (pUC19); Lane 2, DNA + Top1; Lane 3, DNA +Top1 + CPT; Lanes 4, DNA + Top1 + 8l. 9

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Next, compounds 5a and 5b were assayed for antiproliferative activity against A549 (lung cancer), MDA-MB-435 (breast cancer) and HCT116 (colon cancer) cancer cell lines using the MTT assay. Interestingly, the results listed in Table 1 also showed that compound 5b exhibited higher activity against all three cancer cell lines than its diastereoisomer 5a. For the A549 and HCT116 cell lines, compound 5b was about 5-fold more potent than compound 5a with IC50 values of 9.95 µM and 6.35 µM, respectively. The results were consistent with their Top1 inhibitory activity. Although (20S, 21S)-21-fluorocamptothecin was less potent than CPT, it was subjected for structural optimization to improve its antitumor potency.

Table 1 In vitro antitumor activity of 21-fluorocamptothecin diastereoisomers against three cancer cell lines (IC50, µM) Compds

A549

MDA-MB-435

HCT116

5a

46.21

>100

50.91

5b

9.95

58.33

6.35

CPT

0.65

0.45

0.07

Moreover, the hydrolytic stability of active enantiomer 5b and CPT was determined by the HPLC method.28 The results showed that the E-ring of compound 5b was considerably more stable at pH 7.4 than that of natural CPT. For example, after 6 h, 96.0% of compound 5b remained in the original form while only 42.7% of natural CPT remained (Figure 5). To further validate the stability of the designed compounds, compound 8l (a highly active derivative described in the next sections) was also selected for the stability determination. As shown in Figure 5, compound 8l were also 10

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very stable in the PBS buffer, indicating that the α-fluoroether/lactone replacement could significantly improve the hydrolytic stability of CPT E-ring lactone.

Figure 5. The stabilities of CPT, compound 5b and 8l under the condition of pH 7.4.

Antiproliferative Activity of (20S, 21S)-21-Fluorocamptothecin Derivatives A series of (20S, 21S)-21-fluorocamptothecin derivatives 8a-8l with substituents on position 7, 9 or 10 were designed and synthesized to improve antitumor activity and investigate the SARs. To our delight, most of the fluorinated compounds showed potent activities against all the three cancer cell lines (Table 2). Substituents on position 7 of (20S, 21S)-21-fluorocamptothecin played an important role in enhancing antiproliferative activities. For example, compound 8c with ethyl group on position 7 exhibited higher activities than 8b. On the other hand, all the 7-subitituted derivatives showed potent activities against A549 and MDA-MB-435 cells except compound 8g. Notably, antiproliferative activities of compounds 8f, 8j and 8l against A549 were similar to those of CPT. For the MDA-MB-435 cells, compound 8l revealed excellent activity (IC50 = 0.41 µM) while the other compounds showed moderate activities. 11

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Among the fluorinated derivatives, compounds 8f, 8h and 8l were more selective to the HCT116 cell lines with IC50 values of 0.82, 0.27 and 0.07µM, respectively. Particularly,

7-cyclohexyl-21-fluorocamptothecin

(8l)

exhibited

the

best

antiproliferative activity against all the three cancer cell lines (IC50 range: 0.71 µM 0.07 µM), which was 2-fold (A549) and 6-fold (HCT116) more active than CPT. Thus, compound 8l was subjected to a series of cellular and in vivo evaluations.

Table 2 In vitro antitumor activity of (20S, 21S)-fluorocamptothecins against three cancer cells (IC50, µM) Compds

A549

MDA-MB-435

HCT116

8a

11.35

53.02

1.02

8b

41.72

>100

6.15

8c

6.35

12.67

1.45

8d

4.23

8.66

1.46

8e

12.74

10.89

8.53

8f

2.54

2.32

0.82

8g

>100

41.14

65.71

8h

17.70

3.53

0.27

8i

15.46

9.41

3.39

8j

1.00

15.79

95.39

8k

4.14

11.80

19.80

8l

0.71

0.41

0.07

CPT

1.05